Phylogenetic Patterns of Swainsonine Presence in Morning Glories

Endosymbionts play important roles in the life cycles of many macro-organisms. The indolizidine alkaloid swainsonine is produced by heritable fungi that occurs in diverse plant families, such as locoweeds (Fabaceae) and morning glories (Convolvulaceae) plus two species of Malvaceae. Swainsonine is known for its toxic effects on livestock following the ingestion of locoweeds and the potential for pharmaceutical applications. We sampled and tested herbarium seed samples (n = 983) from 244 morning glory species for the presence of swainsonine and built a phylogeny based on available internal transcribed spacer (ITS) sequences of the sampled species. We show that swainsonine occurs only in a single morning glory clade and host species are established on multiple continents. Our results further indicate that this symbiosis developed ∼5 mya and that swainsonine-positive species have larger seeds than their uninfected conspecifics.

INTRODUCTION
Heritable micro-organisms, passed down from mother to offspring through seeds or eggs, play critical roles in the life cycles of many organisms but their prevalence and functional roles are unknown for most plants. This stands in contrast with the well-documented ancient endosymbiotic origins of chloroplasts and mitochondria. Differing from the diverse endophytic fungal communities in plants that result from the environmental transmission and form highly localized infections within single leaves or other tissues (Christian et al., 2015), heritable microorganisms, most often fungal endosymbionts, are associated with only a few selected major plant families. When a vertically transmitted symbiont benefits the host, it has an indirect positive effect on its own fitness as hereditary symbionts are completely dependent on the host for their own propagation and reproductive fitness and so should not reduce host fitness (Ewald, 1987;Lipsitch et al., 1996). Understanding the origin, distribution, and function of heritable fungi provide insights into the evolution and ecology of major plant groups.
Fungi in general produce a wide array of secondary metabolites. The production of bioactive metabolites by endosymbiotic fungi that is observed in multiple hereditary plant-endosymbiotic fungal associations might have particular importance to the fitness of both the fungus and the host species. The benefits and costs of the symbiosis are not well understood in these systems except for the fact that many of these metabolites have toxic effects upon ingestion of the host plant. The indolizidine alkaloid swainsonine (Figure 1) is the toxic principle in a number of plant species worldwide and causes severe toxicosis in livestock grazing these plants (Colegate et al., 1979;Molyneux and James, 1982;Molyneux et al., 1995;de Balogh et al., 1999;Colodel et al., 2002;Dantas et al., 2007). Swainsonine is an alpha-mannosidase and mannosidase II inhibitor that alters glycoprotein processing and causes lysosomal storage disease (Colegate et al., 1979;Dorling et al., 1980;Tulsiani et al., 1988). Consumption of swainsonine-containing plants by grazing animals leads to a chronic disease characterized by weight loss, depression, altered behavior, decreased libido, infertility, and death, which are estimated to cause tens of millions of dollars in livestock losses annually (Panter et al., 1999). Swainsonine occurs sporadically in three diverse plant families: Fabaceae (Fabales), Malvaceae (Malvales), and the morning glory family Convolvulaceae (Solanales) (Cook et al., 2014).
Many of the plant species identified to date that contain swainsonine primarily result from episodes of poisoning as the clinical signs and associated pathology are similar in poisoned livestock (Cook et al., 2014). These cases of livestock poisoning include more than 25 swainsonine-containing legumes commonly referred to as locoweeds (Fabaceae), such as Astragalus, Oxytropis, and Swainsona species. Toxic species within these genera have been identified in Australia, China, North America, and South America. Likewise, seven morning glory species belonging to the genus Ipomoea have been reported to contain swainsonine due to episodes of livestock poisoning in Africa, Australia, and South America (Cook et al., 2014). Lastly, two swainsonine-containing Sida species (Malvaceae) have been identified in South America due to poisoning of livestock (Colodel et al., 2002;Micheloud et al., 2017). More recently, preserved herbarium specimens have been used to test and identify several Astragalus, Oxytropis, and Swainsona species that contain swainsonine (Cook et al., 2016(Cook et al., , 2017b. Herbarium specimens provide an excellent resource to investigate the phytochemical composition of plants and their distribution among species . Prior research has demonstrated that all swainsoninecontaining plant taxa investigated to date are associated with systemic, heritable, seed-transmitted fungal symbionts that produce swainsonine. Swainsonine-containing legumes (Astragalus, Oxytropis, and Swainsona spp.) are associated with a fungal symbiont from Alternaria section Undifilum (Pleosporales) (Braun et al., 2003;Yu et al., 2010;Baucom et al., 2012;Grum et al., 2013). By contrast, the swainsonine-containing convolvulaceous taxon, Ipomoea carnea is associated with a distinct fungal symbiont belonging to the order Chaetothyriales (Cook et al., 2013 and Supplementary Figure 1). Other phylogenetically disjunct groups of swainsonine-producing fungi are found to associate with distantly related plant families. Metarhizium sp. (Hypocreales) are both insect pathogens and plant symbionts while Slafractonia leguminicola (Pleosporales), a pathogen of red clover (Trifolium pratense), also produce swainsonine (Gough and Elliott, 1956;Cook et al., 2014).
Swainsonine-producing fungi that are not associated with plants include several species in the Arthrodermataceae (Onygenales), a family of fungi that causes athlete's foot and ringworm diseases in humans and other mammals (Cook et al., 2017a). Recently, molecular genetic studies have identified and characterized the orthologous swainsonine biosynthetic genes in the above four orders of fungi (Cook et al., 2017a). It is notable that other species of morning glories (Convolvulaceae) are associated with a distinct group of hereditary fungi classified in the genus Periglandula (Clavicipitaceae), that produce the ergot and indole diterpene alkaloids similar to those found in grasses infected with fungal endophytes from the same fungal family (Steiner et al., 2011;Schardl et al., 2013). A recent study found that Periglandula fungi are associated with particular clades of morning glories, where they produce distinct mixtures of ergot alkaloids and where symbiotic hosts have significantly larger seed sizes than non-symbiotic species (Beaulieu et al., 2021).
To further our understanding of the morning glory/Chaetothyriaceae/swainsonine association and to test the hypothesis that the presence of swainsonine, used here to indicate the presence of the heritable symbiont, is found in particular phylogenetic groups, we (1) tested for the presence of swainsonine in seeds of 244 morning glory species from diverse, worldwide herbarium collections in order to determine the global distribution and diversity of Chaetothyriaceae symbiosis, (2) obtained phylogenetically informative internal transcribed spacer (ITS) sequences from GenBank and determined the distribution of swainsonine-positive (S+) and S − species across the morning glory host phylogeny to elucidate the evolutionary history of the symbiosis, and (3) evaluated whether swainsonine and Chaetothyriaceae symbiosis vary with a key plant lifehistory trait to understand potential selective benefits of this symbiosis for the host. Our results provide new insights into the evolution of heritable fungal symbiosis and the diversification of swainsonine-producing fungi in morning glories.

Herbarium Survey
We obtained permissions to sample mature seeds and/or vegetative parts from herbarium specimens from the Missouri Botanical Garden (MOBOT), Vadense Herbarium (WAG, Netherlands), Western Australian Herbarium (PERTH), and the Australian National University Herbarium (ANU), in addition to seeds from personal collections of Convolvulaceae researchers. We sampled mature seeds and/or vegetative parts from a total of 244 morning glory species from 983 herbarium sheets, where species identity, mean swainsonine content, mean seed mass, and mean collection latitude are reported by species for each herbarium specimen where available (Supplementary Data 1). The sampled species include representatives from all continents except Europe (where we had only one sample; Figure 2) and Antarctica. Some species have localized distributions, while others have regional or pantropical distributions (Supplementary Data 1). Our samples represented a subset of all potential host species because most herbarium specimens examined did not have mature seeds. We relied on the records of herbarium specimen labels but realize that misidentifications are possible and have updated any naming conventions to the best of our abilities.

Swainsonine Analysis
Swainsonine was extracted using a modification of a procedure described by Gardner and Cook (2011). A measured quantity of plant material was extracted in a volume of 2% acetic acid for 18 h with agitation. After extraction, samples were centrifuged and an aliquot from the extraction was diluted into 20 mM ammonium acetate in a 1 ml auto-sampler vial. Samples were analyzed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) to detect swainsonine as previously described (Gardner et al., 2001). Presence/absence data were recorded primarily with concentrations being recorded when the sample permitted.
Species that tested positive for swainsonine by the LC-MS/MS method were subsequently verified to contain swainsonine by gas chromatography-mass spectrometry (GC-MS) as a secondary screen. Sample preparation was similar to those described by Cook et al. (2016). All samples were analyzed by GC-MS for swainsonine (trimethylsilyl [TMS] derivative) using the GC-MS conditions previously described (Gardner et al., 2001).

Estimating Morning Glory Phylogeny With ITS Sequence Data
We downloaded the ITS sequence entries from GenBank available in 2021 for 201 species (Supplementary Data 2) across multiple genera (Argyreia, Distimake, Ipomoea, Jacquemontia, Stictocardia, and Merremia) that we sampled for swainsonine. For each species, we obtained the complete ITS1 + 5.8S rRNA + ITS2 sequence where possible. In some cases, this was obtained by combining partial sequences from multiple accessions of the same species. In total, we obtained 194 complete ITS1 + 5.8S + ITS2 sequences, three sequences from combining partial sequences and four were partial ITS1 +5.8S + ITS2 sequences.
These sequences were aligned and trimmed using MAFFT v7.489 (Katoh et al., 2019) using the L-INS-i alignment strategy with a 1.7 gap penalty. We used jModelTest2 (Darriba et al., 2012) on CIPRES Science Gateway (Miller et al., 2010) to determine the best substitution model, which was GTR+G+I based on Akaike Information Criteria (AIC). A maximum-likelihood phylogeny was then estimated using RAxML-HPC2 on XSEDE on CIPRES Science Gateway (Miller et al., 2010) using the GTR+G model, as the developers of RAxML do not recommend using the proportion of invariable sites estimation, with 1,000 bootstrap replicates. We also estimated a Bayesian tree using MrBayes v3.2.7a on XSEDE on CIPRES Science Gateway (Miller et al., 2010) with two runs and four independent chains using the GTR+G+I model for 10,000,000 generations, sampled every 1,000 generations, with the first 25% as burn in. We used Tracer v1.7.2 (Rambaut et al., 2018) to check for convergence. For both methods, we designated Merremia sibirica as the outgroup species based on previous Convolvulaceae phylogenetic work (Simoes et al., 2015).
We recognize the limitations of inferring a phylogeny based on a single gene, but most of our sampled species had no other gene sequences available. Our results are in general agreement with published smaller, multi-gene phylogenies, and the ITS sequences used here have been used to construct phylogenies in other published studies (Muñoz-Rodríguez et al., 2019;Beaulieu et al., 2021).
Testing Phylogenetic Signal for Swainsonine and Seed Mass i) Swainsonine presence: We tested for phylogenetic signal in the presence of swainsonine to determine whether S+ species across the Ipomoeeae tribe are closely related species. We followed the analyses methods described by Beaulieu et al. (2021). In short, we used the function "fitDiscrete" from the R package "geiger" (Pennell et al., 2014) to fit different models of character evolution (FitzJohn et al., 2009). We used the all-rates-different (ARD) model based on the lowest AIC score. We then used Pagel's λ (Pagel, 1999) to assess the phylogenetic signal where there is a strong phylogenetic signal if λ is equal to or near 1. We transformed our phylogeny to one with no phylogenetic signal (λ = 0) and compared its likelihood scores with our observed phylogeny and computed the p-value to determine if our observed phylogeny significantly differed from the nosignal phylogeny.
We also computed Fritz and Purvis's D (FPD) (Fritz and Purvis, 2010) as an alternative measure of the phylogenetic signal using the function "phylo.d" from R package "caper" (Orme et al., 2013). Where D is a measure of phylogenetic dispersion and can be less than 1 (non-random distribution) or equal to 1 (random distribution).
ii) Seed mass: All average seed masses were log10-transformed to normalize the data then phylogenetic signal testing for seed mass was conducted as was done for the presence of swainsonine, except the "fitContinuous" function from the R package "geiger" (Pennell et al., 2014) was used since the data are continuous. The FPD statistic can only be applied to binary traits so we did not apply it to seed mass.

Ancestral Character-State Reconstruction
We followed the analyses methods described by Beaulieu et al. (2021). In short, we reconstructed ancestral states for the presence of swainsonine (yes, no) and seed mass (large, small) using the R packages "ape" (Paradis and Schliep, 2019) and "phytools" (Revell, 2012). We used "ace" from R package "ape" to fit the best model of trait evolution for our data by comparing AIC scores, which show that the ARD model was better than other models (equal-rates and symmetric). This produces a likelihood at each internal node of being S+ or S −, or large or small seeds, character states. Ancestors were designated as being one state or another if the likelihood of it being that state was greater than 75%.

Correlated Evolution in Swainsonine Presence, Concentration, and Seed Mass
We followed the analyses methods described by Beaulieu et al. (2021). In short, we used two methods to test for correlated evolution between swainsonine and seed mass. First, using log10transformed seed mass data, a phylogenetic logistic regression was performed using the function "phyloglm" from the R package "phylolm" (Ho and Ané, 2014). Second, the function "fitPagel" from the R package "phytools" (Revell, 2012) was used to perform Pagel's test of correlation (Pagel, 1994). Pagel's correlation assesses correlated evolution between two binary characters, so we transformed seed mass into a binary trait. The average seed mass of all surveyed species was log10-transformed and was used to separate the large (> 1.615, 41.23 mg) from the small (< 1.615, 41.23 mg) group.
In addition to a linear regression, we use the function "phylolm" from the R package "phylolm" (Ho and Ané, 2014) to perform a phylogenetic linear regression to test for a correlation between seed mass and swainsonine concentration in S+ species (n = 21) where both values were log10-transformed.

Distribution of Swainsonine in Morning Glory Species
In total, 32 of 244 (13%) morning glory species that we evaluated from herbarium specimens (Supplementary Data 1) contained swainsonine and are therefore symbiotic (Figure 2). In total, 24 of the 32 species were not previously known to be S+, and S+ species were distributed across multiple continents ( Table 1). Ipomoea sericosepala was previously reported to be S+ (Mendonça et al., 2018) but we did not detect swainsonine in our samples of the species. Given that there are more than 800 morning glory species, the total number of S+ species will certainly become larger with further sampling.

Swainsonine Distribution in Host Phylogeny
To evaluate the occurrence of swainsonine in relation to morning glory host phylogeny, we obtained published ITS sequences for 201 of our 244 surveyed species (Supplementary Data 2). In both our maximum likelihood and Bayesian inferred phylogenies (Figures 3, 4 and Supplementary Figure 2), we found that all S+ species occurred within a single clade of closely related species and that swainsonine was absent from the rest of the morning glory phylogeny. We found a significant phylogenetic signal for the presence of swainsonine (Pagel's λ = 0.9, D = − 0.67, p < 0.001), suggesting that species with swainsonine are more closely related than expected by chance (Figures 3, 4). Our results suggest that the oldest common ancestor observed was S − and that this symbiosis has arisen relatively recently within the morning glory phylogeny given that the S+ clade was the last to split from the lineage (Figures 3, 4). This pattern also suggests that the symbiosis is strictly heritable and vertically transmitted from a single S+ ancestor, especially given the wide geographic distribution of S+ species. However, several S − species occur within this primarily S+ clade, suggesting that the symbiosis can be lost.

Seed Mass and Swainsonine
Species with larger seeds are often larger as adults, longer-lived, and have lower growth rates than species with smaller seeds (Silvertown, 1981;Moles et al., 2005). Therefore, we measured seed mass as a proxy for plant life history strategy. We found a significant phylogenetic signal in seed mass (λ = 0.89, p < 0.001), suggesting that closely related species have more similar seed mass. Furthermore, seeds of S+ species were approximately two times larger than seeds of S − species (69.5 vs. 37.3 mg, t(32.6) =, p < 0.005, Figure 5A). When we transformed seed mass to a binary state, we found a significantly higher proportion of species with large seeds containing swainsonine than species with small seeds (X 2 = 18.4, p < 0.001, Figure 5B,  Supplementary Figure 3). We then performed Pagel's test for correlated evolution between two discrete characters after accounting for host phylogeny (Pagel, 1994) and found a significant positive correlation between large seed mass and swainsonine (p = 0.047, Figure 4). However, our phylogenetic logistic regression analysis (Ives and Garland, 2010), which tests for a correlation between discrete and continuous characters based on a phylogeny, showed that seed mass as a continuous variable is not a direct predictor of swainsonine presence. Finally, phylogenetic regression (Ho and Ané, 2014) did not show a significant correlation between seed mass and swainsonine concentration in S+ species (Supplementary Figure 4).

DISCUSSION
The results reported here show that all S+ Ipomoea species occur in just a single clade while the rest of the phylogeny is S −. Many of the S+ Ipomoea species reported here are part of Clade A1 described in Wood et al.'s (2020) monograph of Ipomoea species from the Americas. In fact, 22 of 26 species in clade A1 from the Americas surveyed here contained swainsonine. Species in this clade are perennial, generally have tuberous roots, and have distinctive pollen and floral traits (Wood et al., 2020). Within Clade A1, there are additional groups, for example, the Arborescens clade, which is composed of small trees, large shrubs, or lianas, where many are S+ that includes I. pauciflora, I. arborescens, I. chilopsidis, I. intrapilosa, I. populina, I. wolcottiana, and I. murucoides. These common morphological features within this clade might have played a role in the evolution of the swainsonine symbiosis.
This pattern of closely related S+ species observed here is similar to observations in the legume genus Astragalus, which like Ipomoea is species rich and morphologically and phylogenetically complex (Barneby, 1964;Wojciechowski et al., 1999). Unlike the phylogeny of Ipomoea reported by Muñoz-Rodríguez et al. (2019) and herein, the phylogeny of Astragalus generally has poor resolution with only a few well-defined clades due to the recency of the radiation (Wojciechowski et al., 1999). One exception is a phylogenetic clade composed of six North American and eight South American Astragalus species (Wojciechowski et al., 1999;FIGURE 4 | Ancestral state reconstruction of swainsonine presence. Phylogenetic distribution of swainsonine presence and seed mass. Species lacking seed mass data are represented by blank spaces. Pie charts at nodes indicate the relative likelihoods of the swainsonine character state. Scherson et al., 2008). Surveys for S+ species of Astragalus identified swainsonine in 13 of these 14 species in this clade that share a common phylogenetic origin (Cook et al., 2017b,c). Among the more than 50 species of Astragalus that were reported to contain swainsonine in North and South America that includes the 13 mentioned above (Cook et al., 2017b,c), many share common morphological features, such as an inflated pod, as they co-occur in morphologically related taxonomic sections (Barneby, 1964).
Documentation of swainsonine-containing morning glories is relatively recent as compared to other swainsonine-containing legumes (Colegate et al., 1979;de Balogh et al., 1999). Our inferred phylogeny also suggests that the swainsonine symbiosis is a relatively recent plant-fungal association in the Convolvulaceae. We estimate the divergence age of the S+ clade to be approximately 5 mya based on the conclusions of a recently published time-calibrated nuclear phylogeny study of the family (Muñoz-Rodríguez et al., 2019). Further, all S+ species are closely related, which strongly suggest that the fungal endosymbiont is strictly transmitted vertically, consistent with observations on the transmission of the fungal endosymbiont of I. carnea . Furthermore, this pattern suggests that the symbiosis is transmitted vertically during the speciation process. Though S+ species that belong to the same morning glory clade, they are widely distributed geographically across Australia, Africa, Americas, and Southeast Asia, suggesting that these species originated from a common S+ ancestor. The occurrence of occasional S − species within the same clade suggests that the symbiosis has been occasionally lost in the lineage. These S − species also occur over multiple continents. It is highly likely that there are many other Ipomoea species that contain swainsonine given that our samples represent 26 of 127 species (20%) in Clade A1 described by Wood et al. (2020). Based upon our survey reported here, the frequency of S+ species in the clade is greater than 85% or over 100 species.
The Chaetothyriales symbiont associated with I. carnea has been shown to grow as an epibiont on the adaxial leaf surface associated with oil glands , similar to Periglandula on the adaxial surface of Ipomoea asarifolia (Steiner et al., 2011). Plants that lack swainsonine lack the corresponding epibiont on the leaf surface . The epibiont has only been detected growing on the leaf surface and has not been observed growing between cells in different plant tissues. Notably, there is no evidence of horizontal transmission as plants containing the epibiont and plants lacking the epibiont grown in proximity to each other in the greenhouse maintain their original chemical phenotype Noor et al., 2021). It is not known whether the Chaetothyriales symbiont grows as an epibiont in other swainsonine-containing Ipomoea species, but should be examined. In I. asarifolia, Periglandula is associated with and is thought to feed on the oils secreted from the glandular trichomes. Potentially, an analogous type of association may be occurring with the Chaetothyriales symbiont wherein it benefits from the plant through its association with the glandular trichomes thus facilitating the plant host relationship. It is less clear what potential benefits the host plant obtains but theory suggests that there should be a benefit to the host plant.
Interestingly, S − species in the rest of the phylogeny include those that have been shown to contain ergot alkaloids produced by another heritable fungal endosymbiont (Periglandula sp.) found in morning glories, where 25% of species examined contained ergot alkaloids (Beaulieu et al., 2021). Thus, about 40% of all morning glory species examined thus far are symbiotic with one heritable fungus or the other. Swainsonine might affect plant growth in ways that were not captured in our study and this non-overlapping host species distribution of endosymbionts potentially reflects symbiont exclusion to reduce physiological costs to the host or redundancy in functions for the host. A previous study has shown that Periglandulainfected I. tricolor plants have reduced biomass when grown in isolation, but performed better when grown in the presence of root-knot nematodes as compared to non-infected conspecifics (Durden et al., 2019).
Similar to the Periglandula sp. symbiosis in the morning glory family (Beaulieu et al., 2021), S+ species have larger seeds than their S − congeners in our survey. Although the toxic properties of swainsonine can lead to poisoning of animals when ingested, a previous study did not find any evidence that animals avoid S+ plants (Oxytropis and Ipomoea) (Pfister et al., 2020). However, larger seeds represent a larger energy investment by the maternal plant, and swainsonine found in seeds might serve as a deterrent for other potential seed predators, such as birds or insects. For example, Metarhizium sp., which produces toxic metabolites, such as swainsonine, is widely used as an organic plant insecticide (Zimmermann, 2007;St Leger and Wang, 2010). Swainsonine may also increase the fitness of the seed by affecting other fungi associated with the seed or potential seed pathogens. Recent studies have shown that the presence of the heritable symbiont Alternaria fulva and associated swainsonine in the spotted locoweed, Astragalus lentiginosus (Fabaceae), alters the richness and diversity of other fungi associated with the plant (Harrison et al., 2018).
Greater seed mass has also been shown to be characteristic of long-lived plants (Silvertown, 1981) and many of the S+ species here are woody, perennial species (Wood et al., 2020). Larger seeds can increase seedling survival that would be required for the longer time that larger plants take to reach maturity (Moles and Westoby, 2004). Seed mass also correlates with the net primary productivity of the plant and some environmental conditions, such as precipitation (Moles et al., 2005), so it is possible that the correlation of swainsonine present with larger seed mass may reflect other factors than plant life history.
Swainsonine concentrations have been shown to vary between plant tissues, as a function of phenology, and due to endophyte genotype in the legume Oxytropis sericea. In O. sericea, little or no swainsonine was detected in the root or crown of the plant with greater concentrations found in above-ground parts (Cook et al., 2009). Concentrations have been also shown to vary in above-ground parts as a function of developmental age with the greatest concentrations as the plant reaches maturity (Cook et al., 2012). Swainsonine concentrations also vary by endophyte genotype within and between host species, which influences the concentrations found in plant tissues (Braun et al., 2003;Cook et al., 2013). The variation of swainsonine concentrations among different plant tissues and across developmental stages should be examined in different S+ Ipomoea species. Although we did not find a significant correlation between seed mass and swainsonine concentration in S+ species, there is a positive trend that might become stronger with increased sampling (p = 0.075, Supplementary Figure 4). Another interesting pattern is that African S+ species have higher concentrations than S+ species from other continents (Supplementary Figure 4). This group of African species forms a subclade within the S+ clade and their high swainsonine concentrations could suggest a difference in endosymbiont genotypes, consistent with observations in Astragalus and Oxytropis species (Braun et al., 2003;Cook et al., 2013).
Although our study comprised only a fraction (30%) of all described Ipomoea species, it demonstrates the distribution and heritability of swainsonine-producing fungi in morning glory species. The closely related group of S+ morning glories presents a promising future model system for studying heritable fungal symbiosis and its evolution. One important question is whether the diversity and density of other horizontally acquired fungal endophytes in the host microbiome vary with the presence of the heritable symbiont and its swainsonine chemistry as had been shown in other host species (Harrison et al., 2018). In addition, experimental performance and functional studies of S+ vs. S − morning glory species would provide further insights into the costs and benefits of this symbiosis. The genetic diversity and relatedness of the various orders of swainsonineproducing fungal endosymbionts in different plant families also pose interesting directions for studying the origins of these chemically related symbioses, whether these distinct symbioses have similar effects on host plants from different families and fungal-host specificity.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are publicly available. This data can be found here: doi: 10.6084/m9.figshare. 19010885.

AUTHOR CONTRIBUTIONS
DC, QQ, and KC conceptualized the study. DC, DG, and KC collected and generated data. QQ performed statistical analyses with input from KC. All authors contributed to writing, reviewing, and editing the final manuscript.

FUNDING
This study was provided by grant 429440 from the Simons Foundation to the Smithsonian Tropical Research Institute to KC (W. Wcislo, PI).


Haploinsufficiency of the sex-determining genes at MATα restricts genome expansion in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae, mating type of haploid cells is determined by the presence or absence of the MATα idiotype containing MATα1 and MATα2, which encode the transcription factors. These proteins are characterized by rapid turnover, but the physiological relevance of this property remains unclear. Here, we show a direct link between their intracellular levels and sexual stability. Polyploid cells with fewer MATα copies had unstable sexual phenotypes, causing morphological changes and an increase in cell death; these effects were mediated by hyperactivation of the mating pheromone response pathway. Thus, the MATα1 and MATα2 genes are haploinsufficient genes, and the reduction in their product levels causes sex fluctuation. Chromosome III harboring the mating type locus is the most prone to loss in diploids. We propose that the haploinsufficiency of MATα compensates for the drop-out prone nature of chromosome III, thereby suppressing speciation through increased genome size via polyploidization.

INTRODUCTION
Because of its relevance to development, behavior, evolution, and diversity, the mechanism of sex determination has been studied extensively. Sexual dimorphism controlled by the sex-determination system is ubiquitous among eukaryotic organisms from yeasts to humans. Genetic analysis has allowed extensive analysis of the regulation of dichotomous sex and reproduction in the budding yeast Saccharomyces cerevisiae (Herskowitz, 1988). S. cerevisiae haploid cells show either an a-or a-mating type; mating two cells of different mating types produces an a/a diploid. Yeast mating types are determined by the mating type (MAT) locus, which is located on chromosome III and contains either the MATa or MATa idiotype (Klar, 1987). Mata1, which is expressed from MATa, and Mata1 and Mata2, which are expressed from MATa, are transcription factors with multiple targets that give rise to the differences among these three distinct cell types (Galgoczy et al., 2004). MATa contributes to determining haploid mating type, with Mata1 promoting expression of a-specific genes (asg) and Mata2 suppressing a-specific genes (asg) (Strathern et al., 1981). In contrast, MATa is involved in the repression of haploid-specific genes (hsg) in diploid cells but not in the determination of the haploid mating type (Strathern et al., 1981). Therefore, the sexual phenotype (a or a) in haploid yeast is determined by the presence or absence of the MATa (Hanson and Wolfe, 2017).
Because chromosome III is equivalent to sex chromosomes in other eukaryotes, the MAT locus and chromosome III may be involved in speciation, analogous to sex chromosomes in higher eukaryotes (Payseur et al., 2018). Chromosome III is among one of the shortest of 16 chromosomes in S. cerevisiae, and the most prone to loss in diploid yeast (Kumaran et al., 2013). Although an a/a diploid is intrinsically non-mating, it acquires mating ability upon loss of chromosome III, or when a MAT idiotype is mutated or deleted (Haber, 1974). In artificial hybridization by the rare mating of a diploid S. cerevisiae cell with a haploid Saccharomyces kudriavzevii spore/cell, the complete loss of one copy of chromosome III confers mating ability to S. cerevisiae (Morard et al., 2020). It has been speculated that the deletion of one idiotype at the MAT locus is also responsible for whole-genome duplication (WGD), as has occurred in the history of the Saccharomycetales lineage (Marcet-Houben and Gabaldó n, 2015).
Previous studies have shown that the sex-determining MATa products show constant and rapid turnover during vegetative growth, so as not to interfere with mating type switching (Laney and Hochstrasser,

RESULTS
The copy number of sex-determining MATa genes affects polyploid cell morphology and death rate To examine the effects of decreased levels of Mata proteins on the sexual phenotype in polyploid cells, we generated a series of mutants with altered ratios of the MATa copy number to genome set number (M/G ratio). We then examined the effect of the M/G ratio on mortality and morphological parameters such as size, roundness, and slenderness. In MATa polyploid strains with a normal M/G ratio of 1, cell size tended to increase, and cell shape tended to elongate, with increasing ploidy (aa, aaa, and aaaa; Figure 1A). The aaa and aaaa strains were significantly larger and longer (increased by 1.47 and 2.17 times in size and 1.08 and 1.18 times in slenderness, respectively) than the aa strain ( Figure 1B), consistent with the findings of previous polyploidy studies ( Galitski et al., 1999). Interestingly, we found that a decrease in the M/G ratio in MATa polyploid cells caused highly elongated, amorphous Barbapapa-like cell morphologies (DDDa; Figure 1A), which is typical of cells under prolonged exposure to mating pheromones or those that display activation of the abnormal/unsuppressed mating pheromone response pathway (mating pathway) (Siekhaus and Drubin, 2003;Tanaka and Yi, 2009;Udden and Finkelstein, 1978). The DDDa cells showed reduced circularity (0.89-0.77) and were 1.61 times larger and 1.23 times longer than aaaa ( Figure 1B). Diseased cells were apparent in the DDDa culture, as confirmed by selective propidium iodide (PI) staining of dead cells. The PI-positive fraction increased with ploidy, and as the M/G ratio decreased, the latter effect was independent of the ploidy level ( Figure 1C). In comparison, tetraploid cells with complete loss of MATa (DDDD) and only one copy of MATa (aDDD; 6.13 G 2.88%) had a lower percentage of dead cells compared to DDDa cells ( Figures 1A and 1C). These cells were not significantly different from aaaa cells in the morphological parameters ( Figures 1A and 1B). These results are consistent with the observation that matD cells with no MATa behave as an a-mating type (Strathern et al., 1981).
Since a high concentration of mating pheromones can induce rapid cell death (Zhang et al., 2006), we examined the relationship between the mating pathway and mortality in M/G ratio mutants. STE4 encodes the beta-subunit of a heterotrimeric G protein downstream of mating pheromone receptors involved in the mating pathway; receptor signaling is blocked by the loss of Ste4 protein (Hartwell, 1980). In mutants lacking STE4, the morphological changes induced by a reduced M/G ratio were suppressed regardless of ploidy; ste4D M/G ratio mutant strains were comparable to the ploidy-matched MATa polyploid strains in the three parameters ( Figures 1B and 1D), and showed a decreased proportion of PI-positive cells (Figure 1C), indicating that the phenotypes induced by the decreased M/G ratio are dependent on the mating pathway. This result suggests that the DDDa Barbapapa-like morphology is caused by the activation of the mating pathway ( Figure 1A).
Because Mata1 promotes MATa-specific gene expression, whereas Mata2 represses MATa-specific gene expression, we expected that mata1D DDDa and mata2D DDDa would show phenotypes similar to DDDD and DDDa, respectively. Unexpectedly, however, the abnormal morphologies were suppressed in the single mutants ( Figures 1B and 1E). In particular, the induction of mata1D or mata2D resulted in the recovery of circularity (0.77 in DDDa to 0.92 or 0.90, respectively), which was close to the DDDD circularity (0.89). The increased cell death was also suppressed completely by mata1D and partially by mata2D ( Figure 1C). These results indicate that the reduced expression of these two genes is involved in the synthesis of the DDDa phenotype.  Figures 1A, 1D, and 1E. Red bars represent mean values. The calculation of each parameter is described in Quantification and statistical analysis. Different letters represent significant differences, determined by one-way ANOVA and Tukey's HSD comparisons. (C) Fractions of dead cells. The numbers above the graphs are mean values and the error bars indicate the SD(n = 3 experiments). To calculate the p value for M/G ratio mutants with one copy of MATa relative to WT MATa haploid, we applied the Dunnett's test; ***p < 0.001, **p < 0.01. (D) Effect of mata1D and mata2D on the DDDa cell morphology. Scale bar = 20 mm. (E) Effect of ste4D on the cell morphology of M/G ratio mutants. Scale bar = 20 mm.  strasser and Varshavsky, 1990;Nixon et al., 2010). Therefore, we speculated that the different effects of the M/G ratio on the amounts of Mata1 and Mata2 were due to the differences in their respective half-lives.

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Finally, we examined the effect of the M/G ratio using a series of ectopic MATa expressed from loci other than the native MAT. Aberrant morphologies were observed at an M/G ratio of <1, with a lower M/G ratio associated with greater severity of aberrant morphology; the introduction of ectopic MATa reversed the abnormal morphology of M/G ratio mutants ( Figure 3A). For each ploidy, a decrease in roundness and an increase in size and slenderness were observed when the M/G ratio was less than 1 ( Figure 3B). Complementation with ectopic MATa also normalized mortality to the same rate as in the wild type (MATa + 3 3 ectopic MATa tetraploid; 4.92 G 2.99%). These results confirmed that the phenotype of M/G ratio mutants was dependent on the relative copy number of MATa, and indicated that reduced expression of the sex-determining MATa has adverse effects on S. cerevisiae cells.
Cells with a decreased M/G ratio cannot maintain a stable sexual phenotype As described above, the morphology of M/G ratio mutants is associated with the mating pathway. Activation of the mating pathway requires the presence of both mating type cells, or at least the simultaneous expression of a pheromone specific to one mating type cell and its receptor normally expressed in cells of the other mating type. Since the phenotypes of the M/G ratio mutants suggest an abnormality in the sex-determination system, we generated fluorescent reporters that facilitated monitoring the expression of mating type-specific genes. These reporters express green fluorescent protein (GFP) and mCherry downstream of the promoters of MATa-specific MFA1 and MATa-specific MFALPHA1, respectively, which enables mating type discrimination by fluorescence observation. As expected, GFP fluorescence was observed in wild-type (WT) MATa cells, mCherry fluorescence was observed in WT MATa cells, and no fluorescence was observed in WT MATa/MATa diploid cells ( Figure 4A). Moreover, no fluorescence was observed in haploid mata1D cells that lost the expression of both asg and asg (Bender and Sprague, 1989), and fluorescence of both was observed in mata2D cells (which express both asg and asg) (Bender and Sprague, 1989), confirming the efficacy of our fluorescence mating type reporters ( Figure 4A). We found that DDDa emitted not only red fluorescence but also some green fluorescence, although it is genetically MATa ( Figure 4A). This suggests that the M/G ratio mutants cannot stably express either the a-or a-mating type, leading to sex fluctuation.
Our observations with the mating type reporters suggested that not only the pheromone-encoding genes used to make the reporters but also other a-and a-specific genes, such as receptors, were expressed abnormally. We noticed that the mCherry fluorescence of DDDa cells was markedly higher than that of a cells ( Figure 4B), suggesting that the activity of the MFALPHA1 promoter is enhanced in DDDa cells. As the expression of mating pheromone genes was activated by the mating pathway (Roberts et al., 2000;

OPEN ACCESS
Since mating type instability is thought to activate the mating pathway via the binding of mating pheromones to their receptors, we verified the fluorescence intensity of FUS1pr-GFP, using flow cytometry to evaluate the activation level of the mating pathway in the M/G ratio mutants. The FUS1 promoter is commonly used to measure the transcriptional response of the mating pathway (Banderas et al., 2016;Hagen et al., 1991;Paliwal et al., 2007;Takahashi and Pryciak, 2008). Indeed, the M/G ratio mutants had greater FUS1pr-GFP fluorescence, indicating mating pathway activation ( Figure 4C). However, the activity of our FUS1pr-GFP reporter was saturated in mutants with M/G ratios of 1/3 and 1/4. The reporter activity in these mutants was much higher than that of the mata2D and sst2D mutants, which are constitutively activated mating pathway mutants ( Figure 4C) (Bender and Sprague, 1989;Siekhaus and Drubin, 2003). Thus, hyperactivation of the mating pathway explains the high death rate ( Figure 1C) in M/G ratio mutants.
Morphological changes and a high percentage of dead cells in the M/G ratio mutants are not observed after mating with MATa cells Consistent with the mating type instability phenotype ( Figures 4A-4C), M/G ratio mutants showed substantial mating with MATa cells, in addition to normal mating with MATa cells ( Figure 5A). However, no octaploid cells formed when DDDa was cultured alone, under conditions where triploid cells formed by mating in a co-culture of haploid matD cells and haploid MATa cells ( Figure S2), suggesting that the phenotype of M/G ratio mutants mating with MATa cells is unstable. This indicates that the bimater phenotype of M/G ratio mutants is caused by mating type fluctuation, and not solely by chromosome loss, which was reported to occur in polyploid mitosis (Comai, 2005;Kumaran et al., 2013;Mayer and Aguilera, 1990).
Interestingly, M/G ratio mutants showed no morphological changes; despite the increase in ploidy, the DDa and DDDa strains were smaller and shorter (reduced by 0.81 and 0.75 times in size and 0.93 and 0.79 times in slenderness, respectively) after mating with MATa cells ( Figures 5B and S3). Moreover, mating with MATa cells resulted in fewer PI-positive in DDDa cells (tetraploid DDDa; 17.78 G 2.92% to pentaploid aDDDa; 10.81 G 3.71%). The Mata1, Mata1, and Mata2 transcription regulators, which are involved in controlling the expression of mating type-specific genes, are rapidly turned over by degradation of control of the ubiquitin proteasome system (Johnson et al., 1998;Laney and Hochstrasser, 2003;Nixon et al., 2010). By contrast, in the a/a diploid with both MATa and MATa idiotypes formed by mating, Mata1 and Mata2 are stabilized by binding to each other and forming an a1-a2 repressor (Johnson et al., 1998). This supports our model, in which the phenotypes of M/G ratio mutants are due to rapid turnover of Mata proteins normally found in MATa haploid cells, and shows that the expression of Mata proteins with a reduced number of MATa copies is sufficient for function if the product is stabilized after mating.

DISCUSSION
The S. cerevisiae mating type determination system harbors instability Although it was previously thought that the S. cerevisiae mating type was determined by the presence or absence of MATa (Hanson and Wolfe, 2017), we show that the amount of MATa products is also important for sex determination; furthermore, we found that an insufficient quantity of MATa products induces unstable sex-specific gene expression ( Figures 4A and 4B). We speculate that this is due to the rapid turnover of MATa products. In addition to the regulation of Mata proteins turnover described above, it is interesting to note that the expression of MATa1 is repressed in the presence of the a1-a2 repressor (Johnson, 1995). In fact, we observed that, in the a/a-mating type state, a decrease in MATa products was not problematic ( Figures 5B and S3), which supports the idea that this phenomenon is caused by rapid turnover. Nixon et al. (2010) discussed that the rapid turnover prevents simultaneous display of both a-and a-mating  Figure 3A. Red bars represent mean values. The calculation of each parameter is described in Quantification and statistical analysis. The p value was calculated by the Dunnett's test using strains with one copy of MATa in each ploidy as a control group; ***p < 0.001, **p < 0.01, *p < 0.05. iScience Article type phenotypes during mating type switching, and does not interfere with the definition of the new mating type (Nixon et al., 2010). Consistently, pheromone receptors, which are the most upstream factors in the mating pathway, are constitutively taken up by endocytosis (Davis et al., 1993;Jenness and Spatrick, 1986).

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In this study, we demonstrated the impact of decreased MATa products on sexual identity and mating using polyploid cells. We previously observed Barbapapa-like cells when Mata proteins were expressed from MATa on a centromeric plasmid in haploid matD cells (unpublished observation). Since plasmid-borne gene expression is highly variable from cell to cell (Chou et al., 2015), we assume that this phenotype is caused by a shortage of Mata proteins in a fraction of cells with low expression of MATa genes from the plasmid. Thus, we speculate that the ratio of MATa-derived protein amounts to genome size is critical for the stable expression of the sexual phenotype regulated by MATa, irrespective of the ploidy.

Low levels of Mata proteins result in mating pathway confusion
In homothallic strains of S. cerevisiae, switching between the a-and a-mating type phenotypes is completed within the span of a single division cycle, and pheromones and receptors, for example, must also be replaced within this span (Herskowitz, 1988). The mating type reporters used in this study were based on free GFP and mCherry, and their half-lives in yeast cells are longer than the duration of the cell cycle (Mateus and Avery, 2000;Natarajan et al., 1998). This property allows the respective fluorescence emission to continue even after the corresponding gene is repressed. Therefore, our reporters represent sexual history rather than the sex at the moment. Taking advantage of this character, we demonstrated that a decrease in MATa copy number in polyploid cells caused sex fluctuation, as manifested by the expression of asg and asg ( Figures 4A and 4B), and hyperactivation of the mating pathway ( Figure 4B). In addition, hyperactivation was also manifested by strong expression of FUS1pr-GFP ( Figure 4C).
Although DDDa cells exhibited abnormal asg gene expression, they still behaved as the a-mating type, as judged by rare mating with an a-mating type tester ( Figure 5A) and no conspicuous mating in DDDa culture ( Figure S2). Our reporters, which reflect the expression of mating pheromones, provide insight into the specific phenotype of DDDa and the underlying mechanisms by revealing the sexual history. DDDa showed a bias toward mCherry over GFP, i.e., stronger expression of asg than asg ( Figure 4B). Furthermore, our reporter analysis revealed intriguing differences in the expression levels of asg and asg between mata2D and DDDa, although the expression of asg was derepressed in both mutants ( Figures 4A-4C).
When a and a cells are fused to form a zygote, activity of the mating pathway is attenuated through the interaction of MATa-specific Asg7 and MATa-specific Ste3 (Roth et al., 2000). Our flow cytometry data showed that the expression levels of GFP and mCherry in mata2D were comparable to those in MATa and MATa, respectively ( Figure 4B), despite activation of the mating pathway ( Figure 4C). This was presumably mediated by the attenuation of the mating pathway through the interaction of MATa-specific Asg7 and MATa-specific Ste3, which is thought to be important in the zygotic fusion event (Roth et al., 2000) and was proposed to occur in mata2D cells (Rivers and Sprague, 2003). By contrast, DDDa expressed less asg than asg, which would result in insufficient attenuation by Asg7-Ste3, causing hyperactivation of the mating pathway. In conclusion, we assume that DDDa, which is genetically MATa, causes pheromone-receptormediated activation of the mating pathway due to transient expression of the a-mating type phenotype caused by a quantitative deficiency of MATa products.

MATa is composed of haploinsufficient genes encoding transcription factors
The above-described properties are related to the fact that the two proteins expressed from MATa, Mata1 and Mata2, are transcription factors with multiple targets. In cells expressing MATa, Mata1 promotes asg expression and Mata2 represses asg expression, so that the cells stably express the a-mating type (Strathern et al., 1981). It is conceivable that, if the amount of each MATa product is insufficient, the transcriptional iScience Article state of all target genes cannot be maintained. In other words, MATa products have a threshold for the sexual phenotype, and the morphological changes and increased mortality in M/G ratio mutants occurred ( Figures 1A-1C, 3A, and 3B) because it was below this threshold (Figures 2A and 2B).
For Mata2 to act as a transcriptional repressor, it needs to form a complex with Mcm1, which is also involved in cell cycle regulation (Herskowitz, 1989;Keleher et al., 1988). Apart from the prediction that the overproduction of MATa products interferes with mating type switching (Laney and Hochstrasser, 2003;Nixon et al., 2010), we confirmed that MATa2 overexpression causes phenotypes such as growth defects (Figure S4). Since loss-of-function MCM1 mutants have a similar phenotype (Lim et al., 2003), this result may be due to the depletion of Mcm1 by preferential complex formation by excessive Mata2. Taken together with our finding that the phenotype of a MATa polyploid depends on the amount of intracellular Mata ( Figures 1A-1C, 2A, 2B, 3A, 3B, and 4A-4C), these data argue for the need to balance the expression of MATa, neither too much nor too little. This is in line with the dosage-stabilizing hypothesis explaining haploinsufficient (HI) genes, such that a decrease in expression of their products causes a decrease in fitness, while an increase in expression is toxic (Morrill and Amon, 2019). We speculate that MATa has evolved as an HI idiotype. iScience Article HI genes are thought to be ''stuck'' in evolutionary terms, i.e., are unable to regulate gene expression in response to gene dosage variation (Morrill and Amon, 2019). The narrow range of expression of HI genes and significantly reduced fitness when they are outside this range indicate that HI genes are highly sensitive to changes in genome size. The accumulation of HI genes on chromosome III is thought to be the result of selection pressure that prevents diploids from losing this chromosome (de Clare et al., 2011;Delneri et al., 2008). We hypothesize that the haploinsufficiency of MATa, which we discovered, also reduces the fitness of diploids with loss of function of MATa or loss of chromosome III. The loss of a MATa locus in diploids would lead to the production of triploid cells incapable of normal meiosis; S. cerevisiae must have evolved such a mechanism to suppress the emergence of such diploids. In other words, we speculate that the haploinsufficiency of the sexdetermining transcription factor genes at MATa prevents diploids from re-entering the mating stage.
The haploinsufficiency in MATa idiotype suppresses genome expansion Significant events in the genome evolution of S. cerevisiae include WGD and alterations to the sex-determination mechanism (Dujon and Louis, 2017; Hanson and Wolfe, 2017). WGD provides the potential for new functional divergence between paralogous genes and creates a reproductive barrier to ancestral species, leading to speciation (Otto, 2007). WGD is involved in speciation of S. cerevisiae, as indicated by genome comparison between S. cerevisiae and Lachancea waltii (Kellis et al., 2004). As for the mechanism of mating type determination, the transcriptional circuit regulating asg expression of the ancestral species was altered in S. cerevisiae (Hanson and Wolfe, 2017). Mata2, which activates asg expression in ancestral yeast a-mating type cells, was lost in the S. cerevisiae lineage, and S. cerevisiae now uses a Mata2 repressing mechanism of asg expression (i.e., expression asg by default) (Sorrells et al., 2015;Tsong et al., 2003). We speculate that MATa haploinsufficiency evolved after the loss of MATa2 because it depends on the mating type determination mechanism that exhibits an a-mating type by default. It should be noted that the evolution of the asg regulatory circuit is assumed to occur at the same time as WGD, about 100 million years ago (Tsong et al., 2006;Wolfe and Shields, 1997).
Despite experiencing WGD, the current genome of S. cerevisiae is comparable in size to the genome of the pre-WGD yeast L. waltii (approximately 12 and 11 Mb, respectively) (Goffeau et al., 1996;Kellis et al., 2004). This suggests a size-limiting mechanism in the genome of S. cerevisiae, which may be the haploinsufficiency of MATa identified in this study. In conclusion, we speculate that the haploinsufficiency of MATa cooperates with HI genes accumulation on chromosome III to compensate for the loss-prone nature of chromosome III and to suppress speciation with an increased genome size.

Limitations of the study
This study demonstrated that the MATa idiotype is composed of HI genes and that the concentration of their products is involved in the stability of mating type in S. cerevisiae, suggesting that the haploinsufficiency of MATa may affect genome evolution of S. cerevisiae. Although spontaneous diploidization is a relatively common phenomenon under certain stressful conditions and polyploid cells can be easily produced (Galitski et al., 1999;Harari et al., 2018), WGD occurred only once, about 100 million years ago, in the Saccharomycetales lineage (Wolfe and Shields, 1997). We hypothesize that the accumulation of HI genes on the mating type chromosome and the haploinsufficiency of MATa may explain why WGD was an unusual event in the history of the order Saccharomycetales. However, since our research has so far focused exclusively on S. cerevisiae, we have not been able to discuss the evolution of Saccharomycetales. It would be interesting to address the haploinsufficiency of sex-determining genes in species other than S. cerevisiae in a future study.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We thank Drs. Koei Okazaki, Takashi Tsuchimatsu, and Eigo Takeda, and members of Matsuura laboratory for valuable discussions, and Textcheck for editing the manuscript. We also thank anonymous reviewers for critically reading the manuscript and suggesting substantial improvements. This work was supported by JST SPRING, Grant Number JPMJSP2109.

DECLARATION OF INTERESTS
The authors declare no competing interests. d Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

METHOD DETAILS
Yeast strains, plasmids, and media To construct a completely isogenic strain, the mating type of BY4742 was artificially converted from a to a to create OYA38 (BY4742 MATa). First, BY4742 was transformed with YCp50-GAL-HO (Herskowitz and Jensen, 1991), then HO was induced by culturing in medium containing galactose (0.17% yeast nitrogen base without amino acids or ammonium sulfate, 0.5% (NH 4 ) 2 SO 4 , 0.5% casamino acid and 3% galactose (Nacalai Tesque, Kyoto, Japan)) at 30 C for 6 h. Second, the culture was spread on a SDCA plate and formed a single colony. Third, those responsive to a-factor were selected, and then those losing the plasmid were selected in a medium containing 5-fluoroorotic acid.
OYA1 (BY4742 mat::kanMX) was created by transformation of matD with a PCR-based mat::kanMX deletion cassette generated by amplifying kanMX from pFA6a-kanMX (Wach et al., 1994) using primers, MAT del F and MAT del R. Transformants were confirmed by PCR using primers, MAT check F and MAT check R, and sensitivity to a-factor.
MATa/MATa diploid strains were created by rare-mating. To construct a MATa/MATa diploid strain, MATa haploid strains were transformed with plasmids with different auxotrophic markers (pRS313, pRS315) (Baccarini et al., 1983), mixed and cultured overnight on YPD medium plate, and then selected on a SC -Leu -His medium plate in which only the zygote could grow. Then, plasmids were naturally lost by incubation in YPD medium. In the same way, MATa/MATa/MATa triploid strains were constructed by mating a MATa haploid strain and a MATa/MATa diploid strain, and MATa/MATa/MATa/MATa tetraploid strains were constructed by mating MATa/MATa diploid strains with each other. These strains were confirmed to be a-mating type by assessing the formation of sexual agglutination in a co-culture with the MATa tester but not with the MATa tester.
matD/matD diploid strains were created by mating by complementation of matD with MATa-harbored YCpMATa or MATa-harbored YCpMATa plasmid, restoring fertility as an intact a-or a-mating type, respectively. To construct a matD/matD diploid strain, matD strains were transformed with YCpMATa or YCpMATa (Shin et al., 1997) and plasmids with different auxotrophic markers (pRS313, pRS315), mixed and cultured overnight on YPD medium plate, selected on a SC -Leu -His -Ura medium plate in which only the zygote could grow, and then selected for loss of YCpMATa and YCpMATa on 5-FOA medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate, 0.5% (NH 4 ) 2 SO 4 , 0.5% casamino acid, 0.1% 5-fluoroorotic acid, 50 mg/mL uracil and 2% gulcose) plate. Then, cells that lost auxotrophic marker plasmids by incubation in YPD medium were picked up. Similarly, matD/matD/matD triploid strains were created by mating a matD haploid strain and a matD/matD diploid strain, and a matD/matD/matD/ matD tetraploid strain were created by mating matD/matD diploid strains with each other. These strains were confirmed to be a-mating type by assessing the formation of sexual agglutination in a co-culture with the MATa tester but not with the MATa tester.
matD/MATa diploid strains (M/G ratio mutants) were created by mating. To construct a matD/MATa diploid strain, a matD strain with YCpMATa and a MATa haploid strain with pRS313 mixed and cultured overnight on YPD medium plate and selected on a SC -His -Ura medium plate in which only the zygote could grow. Then selected for loss of YCpMATa on 5-FOA medium plate. Then, cells that lost pRS313 by incubation in YPD medium were picked up. Similarly, matD/matD/MATa triploid strains were created by mating a matD/matD diploid strain and a MATa haploid strain, and matD/matD/matD/MATa tetraploid strains were created by mating a matD/matD/matD triploid strain and a MATa haploid strain. These strains were confirmed by PCR using primers, MAT check F and MAT check R, to have two distinct products corresponding to MATa and matD. iScience Article Similarly, the M/G ratio mutant strains shown in Figure 3 were created by mating. First, strains with M/G ratio = 2 (OYA126 MATa ho::MATa), = 3 (OYA280 MATa ho::MATa Chr3::MATa) and = 4 (OYA281 MATa ho::MATa Chr3::MATa Chr14::MATa) were created by sequential introduction of ectopic MATa constructs. Then, the M/G ratio mutant strains were constructed by mating strains having ectopic MATa with each ploidy strain of matD.
In the mating of M/G ratio mutant strains and MATa strain, the strains were also each transformed with plasmids with different auxotrophic markers (pRS313, pRS315), mixed and cultured overnight on YPD medium plate, selected on a SC -Leu -His medium plate in which only the zygote could grow. Then, auxotrophic marker plasmids were naturally lost by incubation in YPD medium.
Since ste4D strains were not capable of mating, pRS317-STE4 was transformed to restore fertility, and ste4D M/G ratio mutants were produced using the method for producing M/G ratio mutants described above.
The mating type reporters are two constructs expressing MFA1 promoter-driven GFP and MFALPHA1 promoter-driven mCherry introduced into intergenic region between SAM50 and SSN8 on Chromosome XIV and HO locus on Chromosome IV, respectively.
To construct cells harboring Chr14::MFA1pr-GFP-kanMX, two PCR fragments were isolated, one amplified with the primers M13U-MFA1P F and NLS-MFA1P R using genomic DNA of BY4742 strain as a template, and the other amplified with the primers NLS-F1 F and Chr14 R using the plasmid pFA6a-GFP(S65T)-kanMX6 (Wach et al., 1997) as a template, resulting the two PCR products were MFA1 promoter and GFP-kanMX sequence. Then, the two resulting fragments were amplified using overlapping PCR with the primers Chr14-M13U F and Chr14 R. The final PCR product was transformed into Chromosome XIV (Location: Chromosome XIV 583,958^583,959) and correct insertion was confirmed by PCR.
The FUS1pr-GFP reporter is a construct expressing FUS1 promoter-driven GFP into HO locus. To construct cells harboring ho::FUS1pr-GFP-kanMX, a fus1::FUS1pr-GFP strain was first constructed through the conventional one-step PCR-based gene replacement. The fus1::FUS1pr-GFP strain was created by replacing the FUS1 ORF of BY4742 with PCR product containing the GFP gene and the kanMX marker gene, using amplified with the primers FUS1pr-F1 F and FUS1 del R and the plasmid pFA6a-GFP(S65T)-kanMX6 as a template. Transformants expressing the GFP from the FUS1 promoter were selected on plates containing G418. Next, to construct a strain in which the HO gene was replaced with FUS1pr-GFP, a fragment was amplified with the primers M13U-FUS1pr F and HO-R1 R using genomic DNA of the fus1::FUS1pr-GFP strain as a template, and then using that fragment as a template, ho::FUS1pr-GFP-kanMX fragment was amplified with the primers HO-M13U F and HO-R1 R. Transformants were selected on plates containing G418 and correct insertion of the fragment was confirmed by PCR.
To quantify Mata1 and Mata2 by Western blotting, Myc and GFP were tagged, respectively. Strains expressing Mata1-13Myc were generated by the conventional one-step PCR-based transformation, using the primers MATa1 Ctag F, MATa1 Ctag R, and the plasmid pFA6a-13Myc-kanMX6 (Longtine et al., 1998). Transformants expressing MATa1-13Myc were selected on plates containing G418. Strains expressing GFP-Mata2 were generated by PCR-based transformation and subsequent marker excision described previously (Khmelinskii et al., 2011), using the primers MATa2 Ntag F and MATa2 Ntag R, and the plasmid pMaM189. First, cells in which the amplified fragment was introduced to express GFP-Mata2 from the NOP1 promoter were selected on SDCA plates. Then, cells which express GFP-MATa2 from the MATa2 promoter were selected in a medium containing 5-fluoroorotic acid. Those selected were cells in which the URA3-NOP1 promoter sequence was popped out by homologous recombination. Correct insertion of the fragment was confirmed by PCR. To construct MATa1 overexpression strains, native MATa1 promoter was replaced with a PCR product containing homologous flanking regions upstream of MATa1 ORF and TEF1 promoter or NOP1 promoter. The PCR products were amplified with the primers MATa1 Ntag F and MATa1 Ntag R, and the plasmid pMaM173 or pMaM189 (Khmelinskii et al., 2011) as a template. Correct insertion of the fragment was confirmed by PCR.
MATa2 overexpression strains were constructed in the same way using the corresponding primers (MATa2 Ntag F and MATa2 Ntag R).
Auxotrophic mutations were rescued by sequential integration of the corresponding wild-type genes as described previously (Maruyama et al., 2016). ura3 and lys2 mutations were complemented by PCR fragments amplified from genomic DNA; URA3 fragments was amplified with the primers URA3 up and URA3 down using genomic DNA of X2180-1A, and LYS2 fragments was amplified with the primers LYS2 up and LYS2 down using genomic DNA of BY4741. his3 and leu2 mutations were complemented by fragments cloned on plasmids; HIS3 fragment was cut out by BamHI and XhoI from pJJ215 (Jones and Prakash, 1990), and LEU2 fragment was cut out by HpaI and SalI from pJJ283 (pJJ252) (Jones and Prakash, 1990). Transformants were then selected by SDCA, SC -Lys, SC -His, or SC -Leu plates.
To construct the plasmid pRS317-STE4, the STE4 fragment was amplified with the primers STE4 GRC F and STE4 GRC R using genomic DNA of BY4742 strain as a template. The fragment was cotransformed into the ste4D strain with HindIII and SacI-digested pRS317 (Sikorski and Boeke, 1991), and the gap-repaired plasmid was then isolated from the transformant.

Microscopy
Yeast cells grown to late log phase in overnight culture were collected by centrifugation (3,000 rpm for 30 s), and then the cells were observed. Fluorescence microscopy was performed by DeltaVision (Applied Precision, Issaquah, WA) and Bright field microscopy was performed by BZ-X810 (Keyence, Osaka, Japan). Images taken in DeltaVision were captured using softWoRx image acquisition and analysis software.
For quantification of cell morphology, overnight culture was stained with FITC-ConA for 30 min, washed with PBS, imaged with BZ-X810, and analyzed with ImageJ.

Analysis of mating type reporters
Yeast cells were grown to log phase and resuspended in PBS. Flow cytometric analysis was conducted with the CytoFLEX S flow cytometer with 488-nm (for GFP) and 561-nm (for mCherry) lasers (Beckman Coulter, Brea, CA). Approximately 10,000 events were acquired for each sample. iScience Article Analysis of FUS1pr-GFP reporter Yeast cells were grown to log phase and resuspended in PBS containing 1 mg/mL Propidium iodide (PI). Flow cytometric analysis was conducted with the CytoFLEX S flow cytometer with 488-nm (for GFP) and 561-nm (for PI) lasers. Dead cells detected by PI staining were excluded, and approximately 10,000 events were acquired for each sample.

Quantification of live/dead cells
Yeast cells were grown to log phase and resuspended in PBS containing 1 mg/mL PI at room temperature for 10 min. Microscopic analysis was conducted with the BZ-X810. At least 200 cells were scored for every strain and the percentage of dead cells stained with PI was calculated.

Mating test
A single colony of each sample strain was picked up with a sterile toothpick and spread as a separate thick vertical strip. On a separate YPD plate, tester strains were spread in the same way. The two plates were incubated at 30 C overnight. By replica plating, strips from the plate of the sample strain were stamped onto a new YPD plate through a sterile velvet cloth. Then, tester strains were transferred to another velvet cloth in the same way, and stamped so that tester strains and sample strains were oriented perpendicular to each other onto the YPD plate that already had sample strains. The plate was incubated overnight at 30 C, then stamped through a sterile velvet cloth into a selective medium in which only zygotes could grow, and incubated at 30 C for 3 days.

Western blot analysis
Protein extraction was performed based on alkaline-trichloroacetic acid method with some modifications. 1.0 OD600 unit of cells was removed from culture, pelleted and stored at À80 C. The pellets were suspended in 200 mL of an alkaline solution (1.85 M NaOH-7.4% 2-mercaptoethanol) and incubated on ice for 10 min. Then, 200 mL of 50% trichloroacetic acid was added and incubated on ice for 10 min. Samples were boiled for 5 min prior to SDS/polyacrylamide gel electrophoresis (SDS/PAGE) and transferred to polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Immunoblot analysis was performed with the indicated antibodies, and the immunoreactive proteins were visualized using ImmunoStar Zeta (Wako Pure Chemical Industries, Ltd.).

DNA content measurement
Flow cytometry analysis method were modified from a previous study (Matsui et al., 2013). Cells were grown overnight and fixed with 70% ethanol at 4 C overnight. Then the cells were washed and suspended in 50 mM sodium citrate, treated with 250 mg/mL RNase A at 50 C for 1 h, and then treated with 1 mg/mL proteinase K at 50 C for 1 h. The resuspended cells were stained with 16 mg/mL propidium iodide at room temperature for 30 min. The DNA content of cells was measured on a CytoFLEX S flow cytometer.

Spot assay
Yeast cells grown to log phase were counted on a haemocytometer and diluted with DW to 1 3 10^7 cells/ ml. The diluted solutions were serially diluted 10-fold and then spotted onto SDCA plate medium. Colonies were photographed after 2 days.